Method and device for performing microoperations on cells

ABSTRACT

A method for performing microoperations on cells consists in that a cell isecured to a micro suction cup and the cell membrane is perforated by a microinstrument. The cell membrane is pierced in a spot under the micro suction cup, the microinstrument being introduced through the recess of the micro suction cup, which is filled with an isotonic solution. 
     A device for performing microoperations on cells comprises a microinstrument (1) installed in a holder (2) rigidly secured to a movable tip (3) a three-way micromanipulator (4), and a micro suction cup (5). The latter is rigidly secured to the holder (2), the microinstrument (1) is located in the recess of the micro suction cup (5) coaxially therewith and is connected to the holder (2) by a means (7a) ensuring the reciprocating movement of the microinstrument (1) along the axis of the micro suction cup (5).

TECHNICAL FIELD

This invention relates to biology and, in particular, to a method forperforming microoperations on cells and a device therefor.

BACKGROUND ART

Known in the art is a method for performing microoperations on cells,comprising the steps of securing a cell by means of a micro suction capand introducing a microinstrument into the cell. The cell is piercedfrom the side opposite to the place where it is secured (cf., forexample, P. Fonbruhn, Metody Mikromanipulyatsii, InostrannayaLiterature, Moscow, pp. 147-149, 1951, in Russian).

The cell membrane is extremely tough and resilient, and cannot bepierced at once by a touch of the sharp tip of the microinstrument, butis punctured only after a substantial deformation of the cell portion tobe pierced and of the whole cell. As a result the cell is seriouslyinjured.

Moreover, large deformation of the cell during the piercing process canlead to membrane rupture and subsequent ejection, and the loss ofcytoplasm due to the general increase of the intracellular pressure. Insome cases even organelles can be displaced in relation to one another.

Ejection of cytoplasm results in upset of the balance between theintracellular matter and the environment, and the cells which have beenoperated on become less viable.

Also known in the art is a method for performing microoperations oncells, which is a method of nuclei transplantation, comprising the stepsof extracting a nucleus from the recipient cell and transplanting itinto the donor cell whose own nucleus had been removed.

This procedure involves two introductions of a microinstrument into acell: the first time to remove the nucleus and the second time to placeanother nucleus. The cell is, therefore, injured twice.

The number of successful operations is, as a rule, rather low (cf., forexample, M. U. Koras, Instrumentation for the Transplantation ofNucleoli, Journal of the Franklin Institute, 262, 407-411, 1956).

Known in the art is a device for performing microoperations on cells,comprising a microinstrument placed in a holder rigidly connected to amobile tool of a three-way micromanipulator, and a micro suction cupfitted on another micromanipulator intended to secure the cell to beoperated on (cf., for example, French Patent No. 1,085,965 published in1965).

This device has a rather complicated system of manipulator control andrelatively low productivity during microoperations, since the operationof two micromanipulators has to be matched. In addition it is expensiveand can still damage the cell when piercing it. The cell is still moredamaged because of the vibration of the micro suction cup andmicroinstrument. Since the cell attached to the micro suction cup issufficiently resilient, the microinstrument can be broken in an attemptto pierce the cell.

DISCLOSURE OF THE INVENTION

The invention is to provide an improved method for performingmicrooperations on cells so as to minimize the deformation of the cellwhen it is perforated and increase the number of successful operations,and also to provide a device to realize this method, which should beconvenient in operation, inexpensive, sufficiently reliable, andefficient enough to cut down the time of microoperations.

There is provided a method for performing microoperations on cells,comprising the steps of securing a cell by means of a micro suction cup,and perforating the cell membrane by a microinstrument, in which,according to the invention, the cell membrane is pierced in the spotunder the micro suction cup, the microinstrument being introducedthrough the recess of the micro suction cup.

The recess of the micro suction cup should be preferably filled with anisotonic solution.

There is also provided a device for performing microoperations on cells,comprising a microinstrument fitted in a holder rigidly connected to amobile tool of a three-way micromanipulator, and a micro suction cup tosecure the cell to be operated on, in which, according to the invention,the micro suction cup is rigidly secured to the holder of themicroinstrument which is located in the recess of the micro suction cupcoaxially therewith and connected to the holder by a device enabling themicroinstrument to move reciprocally along the axis of the micro suctioncup.

The holder should preferably be made as a hollow tee-joint whose onepipe communicates with the recess of the micro suction cup, the oppositepipe features a sealing ring having an opening fitting the diameter ofthe microinstrument, while the third lateral pipe is equipped with anipple in order to produce a negative pressure in the micro suction cuprecess and to provide an input for the isotonic solution.

The microinstrument should preferably be connected to the holder by alengthwise stepping mechanism or a diaphragm box whose case is rigidlysecured to the holder, while the diaphragm is rigidly secured to themicroinstrument, the chamber of the box being communicated with a sourceof control pneumatic impulses.

The method for performing microoperations on cells, according to theinvention, provides for:

elimination of the cell deformation, when a microinstrument pierces thecell;

lesser cell damage;

high precision of introduction of the microinstrument inside the cell toa specific depth;

penetration of the microinstrument into any cell, whatever its shape andsize;

displacement of the cell, if necessary, together with the introducedmicroinstrument;

a single-puncture transplantation of the cell organelles;

microinjection of substances into specific cell portions or individualcell organelles;

a faster microsurgery process.

The device for performing microoperations on cells, according to theinvention, provides, in addition to the advantages inherent in themethod, for high reliability, simplicity of control since only onemicromanipulator is to be operated, a lower price as compared to priorart devices, and less likelihood of breaking a microinstrument whenpiercing the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail with reference tospecific embodiments thereof and accompanying drawings, wherein:

FIG. 1 shows a general view of a device for performing microoperationson cells;

FIG. 2 shows a fragmentary view of a micro suction cup and amicroinstrument with a diaphragm box;

FIG. 3 shows the view of FIG. 1 with a stepping mechanism;

FIG. 4 shows a fragmentary view of an embodiment of a holder for adouble-passage microinstrument;

FIG. 5 shows a schematic of a device for performing microoperations oncells and a cell, as seen in the microscope, when the microinstrument isintroduced into the cell;

FIG. 6 shows a cell, as seen in the microscope, when the microinstrumentis retracted therefrom.

BEST MODE FOR CARRYING OUT THE INVENTION

A method for performing microoperations on cells, according to theinvention, consists in that a cell with is to be subjected tomicrosurgery is secured by means of a micro suction cup. The cell issecured by a negative pressure produced in the recess of the microsuction cup through a microinjector after the cell is brought intocontact with the butt end of the micro suction cup. The cell membrane ispierced by a microinstrument within a spot limited by the butt end ofthe micro suction cup, and the microinstrument is introduced into thecell from the recess of the micro suction cup. The immobilized andstretched-out membrane portion under the micro suction cup is washedwith an isotonic solution which fills the recess of the micro suctioncup prior to the operation. The isotonic solution is introduced into themicro suction cup in order to maintain, at the moment the membrane ispunctured, the balance of the osmotic pressure of the intercellularmatter and the extracellular solution in the zone of the cell membranepuncture. The Ringer solution is used as an isotonic solution for cellsof cold-blooded animals, protozoa and microorganisms, and Ringer-Locke,Ringer-Tirode and other solutions for cells of warm-blooded animals.Calcium iones should be present as the membrane stabilizing agent in allthese solutions so that the damaged portion of the membrane could bequickly restored. In some cases, when transplanting whole intracellularorganelles, for example, where large-diameter microinstruments arerequired and the cell membrane is, therefore, seriously damaged, theisotonic solution contains, as a rule, high-molecular compounds, such ascollidine, dextran and the like, in order to produce a colloidal-osmoticor oncotic pressure which is due to the presence, in the cell cytoplasm,of high-molecular, mainly proteic, substances. All this contributes to afast restoration of the perforated cell membrane and insignificant lossof intracellular components passing into the recess of the micro suctioncup during perforation.

The method for performing microoperations on cells, according to thepresent invention, is remarkable in that the cell is not deformed at allduring perforation because it is held and pierced by a microinstrumentonly in the area of the membrane which is immobilized and stretched outby the tip of the micro suction cup. This portion of the cell membraneis easily perforated by a microinstrument, while the cell is notaffected in any way and is not subjected to deformation.

The use of the proposed method makes the process of cell microsurgerymuch faster since it eliminates an extremely troublesome andtime-consuming search for a perforation spot. Such a spot is determinedby the way the cell is attached to the micro suction cup. Moreover, themicro suction cup and the microinstrument are manipulatedsimultaneously. The microinstrument cannot slide off the membrane duringperforation and is, therefore, introduced into the cell very preciselyirrespective of the cell shape and size.

The method for performing microoperations on cells, according to theinvention, is realized by a device shown in FIGS. 1, 2, 3 and 4.

Referring to FIG. 1, a device for performing microoperations on cells,comprises a microinstrument 1 installed in a holder 2 rigidly connectedwith a movable tip 3 of a three-way micromanipulator 4, and a microsuction cup 5 which is rigidly secured in the holder 2. Themicroinstrument 1 is located in the recess of the micro suction cup 5coaxially therewith and is connected to the holder 2 by a casing 6 and ameans 7a (FIGS. 1 and 2) for reciprocal motion of the microinstrument 1along the axis of the micro suction cup 5.

The holder 2 whose fragmented view is shown in FIG. 2 is a hollowtee-joint 8 whose first pipe 9 is rigidly secured to the micro suctioncup 5 and communicates with the recess of said micro suction cup 5, anopposite pipe 10 houses a sealing ring 11 having a central hole fittingthe diameter of the microinstrument 1, while a third side pipe isequipped with a nipple 12 in order to produce a negative pressure in therecess of the micro suction cup 5 and to provide an input for theisotonic solution.

The means 7a is a diaphragm box comprising a casing 13 and a lid 14, anda diaphragm 15 placed between them and fitted with a plunger 16. Thecasing 13 has a nipple 17 through which the chamber of the diaphragm boxcommunicates with a source of control pneumatic impulses (not shown).The diaphragm 15 is rigidly secured to the microinstrument 1 by theplunger 16 and a clamp 18.

Referring to FIG. 3, an embodiment of a device for performingmicrooperations on cells is provided with a means 7b, which ensures thereciprocating motion of the microinstrument 1 in the recess of the microsuction cup 5 and, unlike the means 7a of the embodiment of FIGS. 1 and2, is a lengthwise stepping mechanism whose casing 19 is rigidly securedto the holder 2 and a movable tip 20 thereof is rigidly secured to themicroinstrument 1.

Referring to FIG. 4 showing a fragmented view of an embodiment of aclamp 18 for a double-passage microinstrument, this clamp 18 comprisessectional pipes 21 and 22 equipped with nipples 23 and 24, respectively,to communicate with respective pipes of the microinstrument 1. The pipes21 and 22 are joined together and sealed by a washer 25. The pipe 21 isalso provided, on its free end, with a sealing washer 26 with a lock-nut27.

The device of FIGS. 1 through 4 operates as illustrated in FIGS. 5 and6, using an example of transplantation of neclei, and goes on asfollows.

Using the micromanipulator 4 (FIG. 1), the microinstrument 1 and themicro suction cup 5 are brought into the field 28 (FIG. 5) of vision ofthe microscope near a cell 29. A separate microinjector connected to thenipple 12 (FIG. 1) is used to provide rarefaction. The tip of the microsuction cup 5 is brought to the cell 29 (FIG. 5) so that it is sucked tothe end of the micro suction cup 5. The portion of the cell membranelimited by the cup is the spot where the microinstrument 1 is introducedinto the cell 29. Then the pressure is supplied through the nipple 17(FIG. 1) into the chamber of the diaphragm box or into the chamber ofthe stepping mechanism 7b (FIG. 3). The diaphragm 15 (FIG. 2) issubjected to deformation and pushes the plunger 16 and the clamp 18holding the microinstrument 1. In the embodiment equipped with astepping mechanism, the movable tip 20 (FIG. 3) pushes themicroinstrument 1. The microinstrument 1 moves forward and perforatesthe membrane portion immobilized and limited by the diameter of themicrosuction cup 5 (FIG. 5). A nucleus 30 taken from another cell is atfirst placed in one of the pipes of the microinstrument 1. In this casethe microinstrument 1 is a double-passage pipette, each passage thereofbeing connected to respective microinjectors which are to suck in andpush out the nuclei. Both operations are performed through themicroinstrument 1 by supplying pressure or rarefaction inside the holder2 (FIG. 4) along two passages through the nipples 23 and 24 (FIG. 4).The isotonic solution which is to reduce the damage effect of the cellmembrane perforation is supplied from a microinjector (not shown)through the nipple 12 (FIG. 5). When the microinstrument 1 is inside thecell 29, a nucleus 31 (FIG. 6) is sucked into the free passage of themicroinstrument 1 and the transplant nucleus 30 which is in the secondpassage of the microinstrument 1 is pushed inside the cell 29. Thismeans that the nuclei transplant operation needs only one perforation tobe completed.

INDUSTRIAL APPLICABILITY

The present invention can be used in the fields of biology, agricultureand medicine. It can be successfully employed for isolation andtransplantation of nuclei and organelles, ovum blastomeres,microinjections of various substances into cells in order to inactivatenuclei, for implantation and incorporation of cell parts or individualcells into other cells or tissues or organisms, for microsurgery ofminute vessels, muscular fibres and other tissues.

We claim:
 1. A method for performing microoperations on cells,consisting in that a cell (29) is secured by means of a micro suctioncup (5) and a microinstrument (1) is used to perforate the membrane ofthe cell (29), characterized in that the membrane of the cell (29) isperforated in a spot under the micro suction cup (5), the instrument (1)being introduced through the recess of the micro suction cup (5).
 2. Amethod for performing microoperations on cells as claimed in claim 1,characterized in that the recess of the micro suction cup (5) is filledwith an isotonic solution.
 3. A device for performing microoperations oncells, comprising a microinstrument (1) installed in a holder (2)rigidly secured to a movable tip (3) of a three-way micromanipulator(4), and a micro suction cup (5) to secure a cell to be perforated,characterized in that the micro suction cup (5) is rigidly secured inthe holder (2), the microinstrument (1) is located in the recess of themicro suction cup (5) coaxially therewith and is connected to the holder(2) by a means (7a and 7b) for reciprocating movement of themicroinstrument (1) along the axis of the micro suction cup (5).
 4. Adevice as claimed in claim 3, characterized in that the holder (2) is ahollow tee-joint (8) whose one pipe (9) communicates with the recess ofthe micro suction cup (5), an opposite pipe (10) houses a sealing ring(11) provides with a hole fitting the diameter of the microinstrument(1), while a third lateral pipe is equipped with a nipple to produce anegative pressure inside the recess of the micro suction cup (5) andprovide an input for the isotonic solution.
 5. A device as claimed inclaim 3, characterized in that the means (7b) connecting themicroinstrument (1) and the holder (2) is a lengthwise steppingmechanism.
 6. A device as claimed in claim 3, characterized in that themeans (7a) connecting the microinstrument (1) and the holder (2) is adiaphragm box whose casing (13) is rigidly secured to the holder (2), adiaphragm (15) is rigidly secured to the microinstrument (1), thechamber of said diaphragm box being communicated with a source ofcontrol pneumatic impulses.